The invention disclosed herein relates to materials and methods for repairing bone defects using osteogenic proteins.
A class of proteins now have been identified that are competent to act as true chondrogenic tissue morphogens, able, on their own, to induce the proliferation and differentiation of progenitor cells into functional bone, cartilage, tendon, and/or ligamentous tissue. These proteins, referred to herein as xe2x80x9costeogenic proteinsxe2x80x9d or xe2x80x9cmorphogenic proteinsxe2x80x9d or xe2x80x9cmorphogens,xe2x80x9d includes members of the family of bone morphogenetic proteins (BMPs) which were initially identified by their ability to induce ectopic, endochondral bone morphogenesis. The osteogenic proteins generally are classified in the art as a subgroup of the TGF-xcex2 superfamily of growth factors (Hogan (1996) Genesand Development 10:1580-1594). Members of the morphogen family of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2), GDF8, GDF9, GDF10, GDF11, GDF12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), the Xenopus homolog Vg1 and NODAL, UNIVIN, SCREW, ADMP, and NEURAL. Members of this family encode secreted polypeptide chains sharing common structural features, including processing from a precursor xe2x80x9cpro-formxe2x80x9d to yield a mature polypeptide chain competent to dimerize, and containing a carboxy terminal active domain of approximately 97-106 amino acids. All members share a conserved pattern of cysteines in this domain and the active form of these proteins can be either a disulfide-bonded homodimer of a single family member, or a heterodimer of two different members (see, e.g., Massague (1990) Annu. Rev. Cell Biol. 6:597; Sampath, et al. (1990) J. Biol. Chem. 265:13198). See also, U.S. Pat. No. 5,011,691; U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085-2093, Wharton et al. (1991) PNAS 88:9214-9218), (Ozkaynak (1992) J. Biol. Chem. 267:25220-25227 and U.S. Pat. No. 5,266,683); (Celeste et al. (1991) PNAS 87:9843-9847); (Lyons et al. (1989 ) PNAS 86:4554-4558). These disclosures describe the amino acid and DNA sequences, as well as the chemical and physical characteristics of these osteogenic proteins. See also Wozney et al. (1988) Science 242:1528-1534); BMP 9 (WO93/00432, published Jan. 7, 1993); DPP (Padgett et al. (1987) Nature 325:81-84; and Vg-1 (Weeks (1987) Cell 51:861-867).
Thus, true osteogenic proteins capable of inducing the above-described cascade of morphogenic events that result in endochondral bone formation have now been identified, isolated, and cloned. Whether naturally-occurring or synthetically prepared, these osteogenic factors, when implanted in a mammal in association with a conventional matrix or substrate that allows the attachment, proliferation and differentiation of migratory progenitor cells, have been shown to induce recruitment of accessible progenitor cells and stimulate their proliferation, thereby inducing differentiation into chondrocytes and osteoblasts, and further inducing differentiation of intermediate cartilage, vascularization, bone formation, remodeling, and finally marrow differentiation. Furthermore, numerous practitioners have demonstrated the ability of these osteogenic proteins, when admixed with either naturally-sourced matrix materials such as collagen or synthetically-prepared polymeric matrix materials, to induce bone formation, including endochondral bone formation under conditions where true replacement bone otherwise would not occur. For example, when combined with a matrix material, these osteogenic proteins induce formation of new bone in: large segmental bone defects, spinal fusions, and fractures. Without exception, each of the above-referenced disclosures describes implantation or delivery of the osteogenic protein at the defect site by packing, filling, and/or wrapping the defect site with an admixture of osteogenic protein and matrix, with the relative volume and surface area of matrix being significant. In the case of non-union defects which do not heal spontaneously, it has heretofore been conventional practice to implant volumes of matrix-osteogenic factor admixtures at the defect site, the volumes being sufficient to fill the defect in order to provide a 3-dimensional scaffold for subsequent new bone formation. While standard bone fractures, can heal spontaneously and without treatment, to the extent the art has contemplated treating fractures with osteogenic proteins, it has been the practice in the art to provide the osteogenic protein together with a matrix locally to a defect site to promote healing.
While implanting a volume of matrix may be conventional wisdom, particularly in the case of non-healing non-union defects, clinical consequences may develop in certain patients as a result of this practice. For example, patients undergoing repeated constructions or defect repairs, or wherein the matrix volume is large, can develop adverse immunologic reactions to matrices derived from collagen. Collagen matrices can be purified, but residual levels of contaminants can remain which is strongly allergenic for certain patients. Alternatively, demineralized autogenic, allogenic or xenogenic bone matrix can be used in place of collagen. Such a matrix is mechanically superior to collagen and can obviate adverse immune reactions in some cases, but proper preparation is expensive, time consuming and availability of reliable sources for bone may be limited. Such naturally-sourced matrices can be replaced with inert materials such as plastic, but plastic is not a suitable substitute since it does not resorb and is limited to applications requiring simple geometric configurations. To date, biodegradable polymers and copolymers have also been used as matrices admixed with osteogenic proteins for repair of non-union defects. While such matrices may overcome some of the above-described insufficiencies, use of these matrices still necessitates determination and control of features such as polymer chemistry, particle size, biocompatability and other particulars critical for operability.
In addition, individuals who, due to an acquired or congenital condition, have a reduced ability to heal bone fractures or other defects that normally undergo spontaneous repair would benefit from methods and injectable compositions that can enhance bone and/or cartilage repair without requiring a surgical procedure. Finally, an injectable formulation also provides means for repairing osteochondral or chondral defects without requiring a surgical procedure.
Needs remain for devices, implants and methods of repairing bone defects which do not rely on a matrix component. Particular needs remain for devices, implants and methods which permit delivery of bone-inducing amounts of osteogenic proteins without concomitant delivery of space-filling matrix materials which can compromise the recipient and/or fail to be biomechanically and torsionally ideal. Needs also remain for providing methods and devices, particularly injectable devices that can accelerate the rate and enhance the quality of new bone formation.
Accordingly, it is an object of the instant invention to provide devices, implants and methods of use thereof for repairing bone defects, cartilage defects and/or osteochondral defects which obviate the need for an admixture of osteogenic protein with matrix. The instant invention provides matrix-free osteogenic devices, implants and methods of use thereof for repairing non-healing non-union defects, as well as for promoting enhanced bone formation for spinal fusions and bone fractures, and for promoting articular cartilage repair in chondral or osteochondral defects. These and other objects, along with advantages and features of the invention disclosed herein, will be apparent from the description, drawings and claims that follow.
The present invention is based on the discovery that an osteogenic or bone morphogenic protein such as OP-1, alone or when admixed with a suitable carrier and not with a conventional matrix material, can induce endochondral bone formation sufficient to repair critical-sized, segmental bone defects. Thus this discovery overcomes the above-described problems associated with conventional materials and methods for repairing bone defects because it permits elimination of matrix material. Furthermore, in view of existing orthopedic and reconstructive practices, this discovery is unexpected and contravenes the art""s current understanding of the bone repair/formation processes.
As disclosed herein, it is now appreciated that an osteogenic protein can be admixed with a carrier as defined herein to form a matrix-free device which, when provided to a mammal, is effective to promote repair of non-union bone defects, fractures and fusions. As disclosed herein, methods and devices are provided for inducing new bone formation at a local defect site without the need for also providing a three-dimensional structural component at the defect site. As contemplated herein, a xe2x80x9cmatrix-freexe2x80x9d osteogenic device is a device devoid of matrix at the time it is provided to a recipient. It is understood that the term xe2x80x9cmatrixxe2x80x9d means a structural component or substrate having a three-dimensional form and upon which certain cellular events involved in endochondral bone morphogenesis will occur; a matrix acts as a temporary scaffolding structure for infiltrating cells having interstices for attachment, proliferation and differentiation of such cells.
The invention provides, in one aspect therefore, a novel method for inducing bone formation in a mammal sufficient to repair a defect. One embodiment comprises the step of providing a matrix-free osteogenic device to a defect locus defining a void. The matrix-free device may be composed of osteogenic protein alone, or it may be composed of osteogenic protein in admixture with a biocompatible, amorphous non-rigid carrier having no defined surfaces. This method induces new bone formation which fills the defect locus, thereby repairing the defect. As contemplated herein, the method comprises providing a matrix-free osteogenic device to a defect locus, wherein the device is provided in a volume insufficient to fill the void at the defect locus. In certain embodiments, the void comprises a volume incapable of endogenous or spontaneous repair. Examples of defects suitable for repair by the instant method include, but are not limited to, critical-sized segmental defects and non-union fractures.
In another embodiment, the invention provides methods and compositions for enhancing fracture repair by providing the matrix-free osteogenic devices described herein to a fracture defect site. The ability of the devices described herein to substantially enhance fracture repair, including accelerating the rate and enhancing the quality of newly formed bone, has implications for improving bone healing in compromised individuals such as diabetics, smokers, obese individuals and others who, due to an acquired or congenital condition have a reduced capacity to heal bone fractures, including individuals with impaired blood flow to their extremities.
In another aspect, the invention provides an implant for inducing bone formation in a mammal sufficient to repair a defect. One preferred implant comprises a matrix-free osteogenic device disposed at a defect locus defining a void. Practice of the above-described method, i.e., providing an osteogenic device devoid of scaffolding structure to a mammal at a defect locus, results in an implant competent to induce new bone formation sufficient to promote repair of non-union bone defects, fractures and fusions. Upon disposition of the osteogenic device at the defect locus, the implant so formed has insufficient volume to fill the defect void.
In yet another aspect, the present invention provides a matrix-free osteogenic device for inducing bone formation in a mammal. As contemplated herein, a preferred osteogenic device comprises an osteogenically-active protein dispersed in a suitable carrier. Preferred osteogenic proteins, include but are not limited to, OP-1, OP-2, BMP-2, BMP-4, BMP-5, and BMP-6 (see below). As disclosed herein, preferred carriers are biocompatible, nonrigid and amorphous, having no defined surfaces or three-dimensional structural features. Thus, the devices of the instant invention lack scaffolding structure and are substantially free of matrix when administered to a mammal. Examples of preferred carriers include, but are not limited to, pluronics and alkylcelluloses. As discussed above, the method of the instant invention involves providing such a device to a defect locus such that the volume of the device is insufficient to fill the void volume at the defect locus.
The methods, implants and devices of the invention also are competent to induce and promote or enhance repair of chondral or osteochondral defects. As a result of this discovery means now are available for promoting bone and/or cartilage repair without requiring a surgical procedure. Particularly as a method for enhancing bone fracture repair, it is contemplated that a suitable formulation can be injected to a fracture site at the time the fracture is set so as to accelerate the rate and enhance the quality of new bone formation.
The device of the instant invention can have a variety of configurations. The nature of the device will be dependent upon the type of carrier in which the osteogenic protein is dispersed. For example, one preferred embodiment can have a paste-like or putty-like configuration; such a device can result from dispersing osteogenic protein in a gel-like carrier such as a xe2x80x9cPluronicxe2x80x9d carrier or an alkylcellulose such as carboxymethyl cellulose which is then wetted with a suitable wetting agent such as, for example, a saline solution. Another preferred embodiment can have a dry powder configuration; such a device results from first dispersing osteogenic protein in a liquid carrier such as water with or without excipient, followed by lyophilization. A third formulation is a solution, such as by combining the protein together with an acidic buffered solution, e.g., pH 4.0-4.5, for example an acetate or citrate buffer. Still another formulation is a suspension formed by disbursing osteogenic protein in a physiologically buffered solution, such as phosphate buffered saline (PBS). Depending upon the configuration of the device, providing it to a defect locus can be accomplished by a variety of delivery processes. For example, a paste can be extruded as a bead which lays along one surface of the defect locus. Alternatively, a viscous liquid can be brushed or painted along one or more surfaces of the defect locus or injected through a wide gauge needle. Less viscous fluids can be injected through a fine gauge needle. Other configurations and modes of delivery are contemplated and discussed below in more detail.
Generally, the proteins of the invention are dimeric proteins that induce endochondral bone morphogenesis. Osteogenic proteins comprise a pair of polypeptides that, when folded, adopt a configuration sufficient for the resulting dimeric protein to elicit a morphogenetic response. That is, osteogenic proteins generally induce all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells. Progenitor cells are uncommitted cells that are competent to differentiate into one or more specific types of differentiated cells, depending on their genomic repertoire and the tissue specificity of the permissive environment in which morphogenesis is induced. In the instant invention, osteogenic proteins can induce the morphogenic cascade which typifies endochondral bone formation.
As used herein, the term xe2x80x9cmorphogenxe2x80x9d, xe2x80x9cbone morphogenxe2x80x9d, xe2x80x9cbone morphogenic proteinxe2x80x9d, xe2x80x9cBMPxe2x80x9d, xe2x80x9costeogenic proteinxe2x80x9d and xe2x80x9costeogenic factorxe2x80x9d embraces the class of proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 are provided in Seq. ID Nos. 1 and 2, respectively. For ease of description, hOP-1 is recited herein below as a representative osteogenic protein. It will be appreciated by the artisan of ordinary skill in the art, however, that OP-1 merely is representative of the TGF-xcex2 subclass of true tissue morphogenes competent to act as osteogenic proteins, and is not intended to limit the description. Other known, and useful proteins include, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, NODAL, UNIVIN, SCREW, ADMP, NURAL and osteogenically active amino acid variants thereof. In one preferred embodiment, the proteins useful in the invention include biologically active species variants of any of these proteins, including conservative amino acid sequence variants, proteins encoded by degenerate nucleotide sequence variants, and osteogenically active proteins sharing the conserved seven cysteine skeleton as defined herein and encoded by a DNA sequence competent to hybridize to a DNA sequence encoding an osteogenic protein disclosed herein. In still another embodiment, useful osteogenic proteins include those sharing the conserved seven cysteine domain and sharing at least 70% amino acid sequence homology (similarity) within the C-terminal active domain, as defined herein.
In still another embodiment, the osteogenic proteins of the invention can be defined as osteogenically active proteins having any one of the generic sequences defined herein, including OPX and Generic Sequences 7 (SEQ ID NO:4) and 8 (SEQ ID NO:5) or Generic Sequences 9 (SEQ ID NO: 8) and 10 (SEQ ID NO:7). OPX accommodates the homologies between the various species of the osteogenic OP1 and OP2 proteins, and is described by the amino acid sequence presented herein below and in Seq. ID No. 3. Generic sequence 9 (SEQ ID NO: 8) is a 102 amino acid sequence containing the six cysteine skeleton defined by hOP1 (residues 330-431 of Seq. ID No. 2) and wherein the remaining residues accommodate the homologies of OP1, OP2, OP3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, UNIVIN, NODAL, DORSALIN, NURAL, SCREW and ADMP. That is, each of the non-cysteine residues is independently selected from the corresponding residue in this recited group of proteins. Generic sequence 10 (SEQ ID NO: 7) is a 97 amino acid sequence containing the seven cysteine skeleton defined by hOP1 (335-431 Seq. ID No. 2) and wherein the remaining residues accommodate the homologies of the above-recited protein group.
As contemplated herein, this family of osteogenic proteins includes longer forms of a given protein, as well as phylogenetic, e.g., species and allelic variants and biosynthetic mutants, including C-terminal addition and deletion mutants and variants, such as those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration still allows the protein to form a dimeric species having a conformation capable of inducing bone formation in a mammal when implanted in the mammal. In addition, the osteogenic proteins useful in this invention may include forms having varying glycosylation patterns and varying N-termini, may be naturally occurring or biosynthetically derived, and may be produced by expression of recombinant DNA in procaryotic or eucaryotic host cells. The proteins are active as a single species (e.g., as homodimers), or combined as a mixed species, including heterodimers.
The methods and implants of the invention do not require a carrier for the osteogenic protein to induce bone formation sufficient to fill a critical size bone defect or to enhance fracture repair in an animal. When the protein is provided in association with a carrier in the practice of the invention, the carrier must lack a scaffolding structure, as stated above. When a preferred carrier is admixed with an osteogenic protein, a device is formed which is substantially free of matrix as defined herein. xe2x80x9cSubstantially free of matrixxe2x80x9d is understood to mean that, the carrier-containing device as formulated prior to administration, does not contain a substrate competent to act as a scaffold per se. That is, the device contains no substrate which has been introduced from an exogenous source and is competent to act as a scaffold. Stated another way, prior to delivery, the carrier is recognized, by virtue of its chemical nature, to be unable to contribute a scaffolding structure to the device. By definition, preferred carriers are biocompatible, non-rigid and amorphous, having no defined surfaces. As used herein, xe2x80x9cnon-rigidxe2x80x9d means a carrier formulation that is lax or pliant or otherwise is substantially incapable of providing or forming a three-dimensional structure having one or more defined surfaces. As used herein, xe2x80x9camorphousxe2x80x9d means lacking a definite three-dimensional form, or specific shape, that is, having no particular shape or form, or having an indeterminate shape or form. Preferred carriers are also biocompatible, non-particulate, adherent to bone, cartilage and/or muscle, and inert. In certain embodiments, water-soluble carriers are preferable. Additionally, preferred carriers do not contribute significant volume to a device of the instant invention. That is, a preferred carrier permits dispersal of an osteogenic protein such that the final volume of the resulting device is less than the volume of the void at the defect locus. As discussed below, a preferred carrier can be a gel, an aqueous solution, a suspension or a viscous liquid. For example, particularly preferred carriers can include, without limitation, poloxamers, alkylcelluloses, acetate buffers, physiological saline solutions, lactose, mannitol and/or other sugars. Alternatively, osteogenic proteins can be provided alone to a defect site.
In summary, the methods, implants and devices of the present invention can be used to induce endochondral and intramembranous bone formation sufficient to repair bone defects which do not heal spontaneously, as well as to promote and enhance the rate and/or quality of new bone formation, particularly in the repair of fractures and fusions, including spinal fusions. The methods, implants and devices also are competent to induce repair of osteochondral and/or subchondral defects. That is, the methods, implants and devices are competent to induce formation of new bone and the overlying surface cartilage. The present invention is particularly suitable for use in collagen- or matrix-allergenic recipients. It is also particularly suitable for use in patients requiring repetitive reconstructive surgeries, as well as cancer patients as an alternative to reconstructive procedures using metal joints. The present invention also is useful for individuals whose ability to undergo spontaneous bone repair is compromised, such as diabetics, smokers, obese individuals, immune-compromised individuals, and any individuals have reduced blood flow to their extremities. Other applications include, but are not limited to, prosthetic repair, spinal fusion, scoliosis, cranial/facial repair, and massive allograft repair.
In order to more clearly and concisely describe the subject matter of the claimed invention, the following definitions are intended to provide guidance as to the meaning of specific terms used in the following written description and appended claims.
xe2x80x9cBone formationxe2x80x9d means formation of endochondral bone or formation of intramembranous bone. In humans, bone formation begins during the first 6-8 weeks of fetal development. Progenitor stem cells of mesenchymal origin migrate to predetermined sites, where they either: (a) condense, proliferate, and differentiate into bone-forming cells (osteoblasts), a process observed in the skull and referred to as xe2x80x9cintramembranous bone formation;xe2x80x9d or, (b) condense, proliferate and differentiate into cartilage-forming cells (chondroblasts) as intermediates, which are subsequently replaced with bone-forming cells. More specifically, mesenchymal stem cells differentiate into chondrocytes. The chondrocytes then become calcified, undergo hypertrophy and are replaced by newly formed bone made by differentiated osteoblasts which now are present at the locus. Subsequently, the mineralized bone is extensively remodeled, thereafter becoming occupied by an ossicle filled with functional bone-marrow elements. This process is observed in long bones and referred to as xe2x80x9cendochondral bone formation.xe2x80x9d In postfetal life, bone has the capacity to repair itself upon injury by mimicking the cellular process of embryonic endochondral bone development. That is, mesenchymal progenitor stem cells from the bone-marrow, periosteum, and muscle can be induced to migrate to the defect site and begin the cascade of events described above. There, they accumulate, proliferate, and differentiate intocartilage which is subsequently replaced with newly formed bone.
xe2x80x9cDefectxe2x80x9d or xe2x80x9cdefect locusxe2x80x9d as contemplated herein defines a void which is a bony structural disruption requiring repair. The defect further can define an osteochondral defect, including both a structural disruption of the bone and overlying cartilage. xe2x80x9cVoidxe2x80x9d is understood to mean a three-dimensional defect such as, for example, a gap, cavity, hole or other substantial disruption in the structural integrity of a bone or joint. A defect can be the result of accident, disease, surgical manipulation and/or prosthetic failure. In certain embodiments, the defect locus is a void having a volume incapable of endogenous or spontaneous repair. Such defects are also called critical-sized segmental defects. The art recognizes such defects to be approximately 3-4 cm, at least greater than 2.5 cm, gap incapable of spontaneous repair. In other embodiments, the defect locus is a non-critical segmental defect approximately at least 0.5 cm but not more than approximately 2.5 cm. Generally, these are capable of some spontaneous repair, albeit biomechanically inferior to that made possible by practice of the instant innovation. In certain other embodiments, the defect is an osteochondral defect such as an osteochondral plug. Other defects susceptible to repair using the instant invention include, but are not limited to, non-union fractures; bone cavities; tumor resection; fresh fractures; cranial/facial abnormalities; spinal fusions, as well as those resulting from diseases such as cancer, arthritis, including osteoarthritis, and other bone degenerative disorders. xe2x80x9cRepairxe2x80x9d is intended to mean induction of new bone formation which is sufficient to fill the void at the defect locus, but xe2x80x9crepairxe2x80x9d does not mean or otherwise necessitate a process of complete healing or a treatment which is 100% effective at restoring a defect to its pre-defect physiological/structural state.
xe2x80x9cMatrixxe2x80x9d is understood in the art to mean an osteoconductive substrate having a scaffolding structure on which infiltrating cells can attach, proliferate and participate in the morphogenic process culminating in bone formation. In certain embodiments, matrix can be particulate and porous, with porosity being a feature critical to its effectiveness in inducing bone formation, particularly endochondral bone formation. As described earlier, a matrix is understood to provide certain structural components to the conventional osteogenic device (i.e., heretofore comprising a porous, particulate matrix component such as collagen, demineralized bone or synthetic polymers), thereby acting as a temporary and resorbable scaffolding structure for infiltrating cells having interstices for attachment, proliferation and differentiation of such cells. Accordingly, the term xe2x80x9cmatrix-free osteogenic devicexe2x80x9d or an osteogenic device which is xe2x80x9csubstantially free of matrixxe2x80x9d contemplates a device which is devoid of an art-recognized matrix at the time it is provided to a recipient. Moreover, substantially free of matrix is understood to mean that, when a device is provided to a defect locus, no substrate competent to act as a scaffold per se is introduced from an exogenous source. Matrix-free or substantially free of matrix is not intended to exclude endogenous matrix which is induced or formed following delivery of the devices and/or implants disclosed herein to a defect locus. Thus the present invention further contemplates a method of inducing endogenous matrix formation by providing to a defect locus the matrix-free devices or implants disclosed herein.
xe2x80x9cOsteogenic devicexe2x80x9d is understood to mean a composition comprising osteogenic protein dispersed in a biocompatible, non-rigid amorphous carrier having no defined surfaces. Osteogenic devices of the present invention are competent to induce bone formation sufficient to fill a defect locus defining a void. Osteogenic devices are matrix-free when provided to the defect locus and are delivered to the defect locus in a volume insufficient to fill the void defined by the defect locus. A device can have any suitable configuration, such as liquid, powder, paste, or gel, to name but a few. Preferred properties of osteogenic devices suitable for use with the method of the instant invention include, but are not limited to: adherent to bone, cartilage and/or muscle; and, effective to provide at least a local source of osteogenic protein at the defect locus, even if transient. As contemplated herein, providing a local source of protein includes both retaining protein at the defect locus as well as controlled release of protein at the defect locus. All that is required by the present invention is that the osteogenic device be effective to deliver osteogenic protein at a concentration sufficient to induce bone formation that fills the three-dimensional defect defining the void requiring repair. In addition to osteogenic proteins, various growth factors, hormones, enzymes therapeutic compositions, antibiotics, or other bioactive agents can also be contained within an osteogenic device. Thus, various known growth factors such as EGF, PDGF, IGF, FGF, TGF-xcex1, and TGF-xcex2 can be combined with an osteogenic device and be delivered to the defect locus. An osteogenic device can also be used to deliver chemotherapeutic agents, insulin, enzymes, enzyme inhibitors and/or chemoattractant/chemotactic factors.
xe2x80x9cOsteogenic proteinxe2x80x9d or bone morphogenic protein is generally understood to mean a protein which can induce the full cascade of morphogenic events culminating in endochondral bone formation. As described elsewhere herein, the class of proteins is typified by human osteogenic protein (hOP 1). Other osteogenic proteins useful in the practice of the invention include osteogenically active forms of OP 1, OP2, OP3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, DPP, Vg1, Vgr, 60A protein, GDF-1, GDF-3, GDF-5, 6, 7, BMP10, BMP11, BMP13, BMP15, UNIVIN, NODAL, SCREW, ADNT or NURAL and amino acid sequence variants thereof. In one currently preferred embodiment, osteogenic protein include any one of: OP1, OP2, OP3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acid sequence variants and homologs thereof, including species homologs, thereof. Particularly preferred osteogenic proteins are those comprising an amino acid sequence having at least 70% homology with the C-terminal 102-106 amino acids, defining the conserved seven cystein domain, of human OP-1, BMP2, and related proteins. Certain preferred embodiments of the instant invention comprise the osteogenic protein, OP-1. Certain other preferred embodiments comprise mature OP-1 solubilized in a physiological saline solution. As further described elsewhere herein, the osteogenic proteins suitable for use with Applicants"" invention can be identified by means of routine experimentation using the art-recognized bioassay described by Reddi and Sampath. xe2x80x9cAmino acid sequence homologyxe2x80x9d is understood herein to mean amino acid sequence similarity. Homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or allowed point mutations of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to or are conservative substitutions of the corresponding residues in a reference sequence. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term xe2x80x9cconservative variationxe2x80x9d also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
Proteins useful in this invention include eukaryotic proteins identified as osteogenic proteins (see U.S. Pat. No. 5,011,691, incorporated herein by reference), such as the OP-1, OP-2, OP-3 and CBMP-2 proteins, as well as amino acid sequence-related proteins such as DPP (from Drosophila), Vg1 (from Xenopus), Vgr-1 (from mouse), GDF-1 (from humans, see Lee (1991), PNAS 88:4250-4254), 60A (from Drosophila, see Wharton et al. (1991) PNAS 88:9214-9218), dorsalin-1 (from chick, see Basler et al. (1993) Cell 73:687-702 and GenBank accession number L12032) and GDF-5 (from mouse, see Storm et al. (1994) Nature 368:639-643). BMP-3 is also preferred. Additional useful proteins include biosynthetic morphogenic constructs disclosed in U.S. Pat. No. 5,011,691, e.g., COP-1, 3-5, 7 and 16, as well as other proteins known in the art. Still other proteins include osteogenically active forms of BMP-3b (see Takao, et al., (1996), Biochem. Biophys. Res. Comm. 219: 656-662. BMP-9 (see WO95/33830), BMP-15 (see WO96/35710), BMP-12 (see WO95/16035), CDMP-1 (see WO94/12814), CDMP-2 (see WO94/12814), BMP-10 (see WO94/26893), GDF-1 (see WO92/00382), GDF-10 (see WO95/10539), GDF-3 (see WO94/15965) and GDF-7 (WO95/01802).
Still other useful proteins include proteins encoded by DNAs competent to bybridize to a DNA encoding an osteogenic protein as described herein, and related analogs, homologs, muteins and the like (see below).
xe2x80x9cCarrierxe2x80x9d as used herein means a biocompatible, non-rigid, amorphous material having no defined surfaces suitable for use with the devices, implants and methods of the present invention. As earlier stated, xe2x80x9cnon-rigidxe2x80x9d means a carrier formulation that is lax or pliant or otherwise is substantially incapable of providing or forming a three-dimensional structure having one or more defined surfaces. As used herein, xe2x80x9camorphousxe2x80x9d means lacking a definite three-dimensional form, or specific shape, that is, having no particular shape or form, or having an indeterminate shape or form. Suitable carriers also are non-particulate and are non- porous, i.e., are pore-less. Carriers suitable for use in the instant invention lack a three-dimensional scaffolding structure and are substantially matrix-free. Thus, xe2x80x9csubstantially free of matrixxe2x80x9d is also understood to mean that, when a carrier-containing device is provided to a defect locus, no substrate competent to act as a scaffold per se is introduced from any exogenous source, including the carrier. Prior to delivery to and implantation in the recipient, the carrier is recognized by virtue of its chemical nature to be unable to contribute a three-dimensional scaffolding structure to the device. Preferred carriers are adherent, at least transiently, to tissues such as bone, cartilage and/or muscles. Certain preferred carriers are water-soluble, viscous, and/or inert. Additionally, preferred carriers do not contribute significant volume to a device. Currently preferred carriers include, without limitation alkylcelluloses, poloxamers, gelatins, polyethylene glycols, dextrins, vegetable oils and sugars. Particularly preferred carriers currently include but are not limited to poloxamer 407, carboxymethylcelluloses, lactose, mannitol and sesame oil. Other preferred carriers include acetate buffer (20 mM, pH 4.5), physiological saline (PBS), and citrate buffer. In the case of devices comprising carriers such as acetate, poloxamers and PBS, administration by injection can result in precipitation of certain osteogenic proteins at the administration site.
xe2x80x9cImplantxe2x80x9d as contemplated herein comprises osteogenic protein dispersed in a biocompatible, nonrigid amorphous carrier having no defined surfaces disposed at a defect locus defining a void. That is, the implant of the present invention is contemplated to comprise the defect locus per se into/onto which the device of the present invention has been delivered/deposited. It is further contemplated that, at the time of delivery of a device, an implant lacks scaffolding structure and is substantially matrix-free. Implants resulting from practice of the instant method are competent to induce bone formation in a defect locus in a mammal sufficient to fill the defect with newly formed bone without also requiring inclusion of a matrix or scaffolding structure, at the time of delivery of a device, sufficient to substantially fill the void thereby structurally defining the defect size and shape.